TiO₂ micro-nano-hybrid surface to alleviate biological aging of UV-photofunctionalized titanium

Figures & data

Figure 1 TiO₂ nanonodules in a tailored size of 300 nm created within micropit architecture of titanium surfaces. (A) The strategic design of micro-nano-hybrid topography of TiO₂ is described schematically. Addition of 300 nm nanonodules to the acid-etching-created micropit surface was planned. (B) Scanning electron micrography (SEM) before and after TiO₂ sputter deposition onto the acid-etched (micropit) surface. Note that the nanonodular development of TiO₂ at the flank (arrowheads) and top of existing peaks, while preserving the peak lines of the microscale compartments, manifesting the hybrid configuration of micro- and nano-architecture as a result. (C) Quantitative measurement of surface roughness and surface area of the micropit surface with and without 300 nm nanonodules (n = 9). The surface area was measured in a 5 μm × 5 μm horizontal plane.

Notes: ^aP < 0.05; ^bP < 0.01, indicating a statistically significant difference between the surfaces with micropits alone and with micropits and nanonodules.

Figure 2 Age-dependent changes in hydrophilicity of micropit and micro-nano-hybrid titanium surfaces after UV treatment. Fresh (immediately after UV treatment) and aged (3 and 7 days after UV treatment) UV-treated titanium surfaces as well as UV-untreated titanium surfaces are compared. Upper panels show the top and side views of titanium discs immediately after a 10 μL droplet of distilled H₂O was placed. Lower panels show the calculated area of spread and the contact angle of H₂O.

Note: Data are mean ± SD (n = 3).

Figure 3 Age-dependent change of osteoblast attachment to UV-treated micropit and micro-nano-hybrid titanium surfaces. UV-treated titanium disks of different ages – fresh (immediately after UV treatment), 3-day-old (stored for 3 days in the dark after UV treatment) 0, and 7-day-old surfaces – were tested. Titanium disks before UV treatment are also compared. The number of rat bone marrow–derived osteoblasts attached to titanium surfaces after incubation for 3 hours ([A] early stage attachment) and 24 hours ([B] late-stage attachment), as evaluated by WST-1 colorimetry.

Notes: Data are mean ± SD (n = 3); ^aP < 0.05; ^bP < 0.01, indicating a statistically significant effect of age of titanium.

Figure 4 Age-dependent change of osteoblast affinity of UV-treated titanium surfaces evaluated by the initial spread and cytoskeletal arrangement of osteoblasts. Osteoblasts were seeded onto UV-treated micropit and micro-nano-hybrid surfaces of different ages – fresh (immediately after UV treatment) and 7-day-old surfaces. Representative confocal microscopic images of cells stained with rhodamine phalloidin for actin filaments (red) and antivinculin (green) 6 hours after seeding are presented. Low (A) and high (B) magnification images, along with cytomorphometric evaluations (C), are presented.

Notes: Data are mean ± SD for panel C (n = 10); ^aP < 0.05; ^bP < 0.01, indicating a statistically significant effect of age of titanium.

Figure 5 Osteoblast proliferation on differently aged UV-treated titanium surfaces. Osteoblasts were cultured on UV-treated micropit and micro-nano-hybrid titanium disks with different ages – fresh, 3-day-old, and 7-day-old surfaces. (A) Cell density at day 3 of culture. (B) Cell proliferative activity evaluated by BrdU incorporation per cell at day 3 of culture.

Notes: Data are mean ± SD (n = 3) for all panels; ^aP < 0.01, indicating a statistically significant effect of age of titanium.

Figure 6 Osteoblast functions on differently aged UV-treated titanium surfaces. Osteoblasts were cultured on UV-treated micropit and micro-nano-hybrid titanium disks with different ages – fresh, 3-day-old, and 7-day-old surfaces. (A) Alkaline phosphatase activity at day 7. (B) Calcium deposition at day 14 of culture.

Notes: Data are mean ± SD (n = 3) for all panels; ^aP < 0.05; ^bP < 0.01, indicating a statistically significant effect of age of titanium.

Figure 7 Experimental results addressing the mechanism underlying the sustained high bioactivity of UV-treated micro-nano-hybrid titanium surfaces. Osteoblast attachment after 3 hours (early stage attachment [A] and 24 hours late-stage attachment, [B] incubation plotted against the H₂O contact angle on each of micropit and micro-nano-hybrid surfaces in various ages after UV treatment, showing no significant correlation between them). (C) Electrostatic effects on osteoblast attachment capability of titanium surfaces. UV-treated micropit and micro-nano-hybrid surfaces with different age (fresh and 7-day-old) were tested for cell attachment capability with and without an additional treatment of the surfaces with NaCl solution.

Notes: Data are mean ± SD (n = 3); ^aP < 0.01, indicating a statistically significant difference between NaCl-treated and untreated surfaces.

Figure 8 Age-dependent change of osteoblast attachment to UV-treated micropit surfaces with 50-nm-thin TiO₂ coating. (A) SEM images of the 50-nm TiO₂ coated micropit surfaces. The number of rat bone marrow–derived osteoblasts attached to titanium surfaces after incubation for 3 hours ([A] early stage attachment) and 24 hours ([B] late-stage attachment), as evaluated by WST-1 colorimetry.

Notes: Data are mean ± SD (n = 3); UV-treated titanium disks of different ages – fresh (immediately after UV treatment), 3-day-old (stored for 3 days in the dark after UV treatment), and 7-day-old surfaces – were tested; the data from micro-nano-hybrid surfaces (300 nm nodules in micropits) are also presented; ^aP < 0.05, indicating a statistically significant effect of age of titanium.